RESUMEN
BACKGROUND: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. METHODS: SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. RESULTS: The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. CONCLUSIONS: The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. GENERAL SIGNIFICANCE: Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Neoplasias de la Mama/metabolismo , Clatrina/metabolismo , Doxorrubicina/metabolismo , Portadores de Fármacos , Endocitosis , Ácido Fólico/metabolismo , Magnetismo/métodos , Nanopartículas de Magnetita , Nanomedicina/métodos , Polietilenglicoles/química , Neoplasias del Cuello Uterino/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Caveolas/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Doxorrubicina/química , Doxorrubicina/farmacología , Endosomas/metabolismo , Femenino , Ácido Fólico/química , Células HeLa , Humanos , Lisosomas/metabolismo , Células MCF-7 , Nanopartículas de Magnetita/química , Microscopía Confocal , Microscopía Electrónica de Transmisión de Rastreo , Microscopía de Fluorescencia por Excitación Multifotónica , Espectroscopía de Pérdida de Energía de Electrones , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
The anti-tumour effect of ferrociphenol (FcdiOH)-loaded lipid nanocapsules (LNCs), with or without a DSPE-mPEG2000 coating, was evaluated on an orthotopic gliosarcoma model after administration by convection-enhanced delivery (CED) technique or by intra-carotid injection. No toxicity was observed by MRI nor by MRS in healthy rats receiving a CED injection of FcdiOH-LNCs (60µL, 0.36mg of FcdiOH/rat) when the pH and osmolarity had been adjusted to physiological values prior to injection. At this dose, the treatment by CED with FcdiOH-LNCs significantly increased the survival time of tumour-bearing rats in comparison with an untreated group (28.5 days vs 25 days, P=0.0009) whereas DSPE-mPEG2000-FcdiOH-LNCs did not exhibit any efficacy with a median survival time of 24 days. After intra-carotid injection (400µL, 2.4mg of FcdiOH/rat), hyperosmolar DSPE-mPEG2000-FcdiOH-LNCs markedly increased the median survival time (up to 30 days, P=0.0008) as compared to the control (20%). This was strengthened by their evidenced accumulation in the tumour zone and by the measure of the fluorescent brain surface obtained on brain slides for these DiI-labelled LNCs, being 3-fold higher than for the control. These results demonstrated that, depending upon the administration route used, the characteristics of LNC suspensions had to be carefully adapted.